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Co-culture with Mature Islet Cells Augments the Differentiation of Insulin-Producing Cells from Pluripotent Stem Cells

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Abstract

Islet transplantation has been hampered by the shortage of islet donors available for diabetes therapy. However, pluripotent stem cells (PSCs) can be an alternative source of insulin-producing cells (IPCs) because of their capacity for self-renewal and differentiation. We described a method to efficiently differentiate PSCs into IPCs by co-culturing mature islets with directed-differentiated pancreatic endoderm (PE) cells from mouse and human PSCs. PE cells co-cultured with islet cells or islet cell-derived conditioned medium (CM) showed increased expression levels of β-cell markers; significantly higher levels of proinsulin- and Newport Green (NG)-positive cells, which revealed the characteristics of insulin producing cells; and increased insulin secretion upon glucose stimulation. Co-culturing human PE cells with islet cells was also effective to differentiate PE cells into IPCs. Diabetic nude mice transplanted with co-cultured cells exhibited restored euglycemia, human C-peptide release, and improved glucose tolerance. Immunohistochemistry revealed that insulin+/C-peptide + cells existed in the grafted tissues. These results suggest that mature islet cells can increase the differentiation efficiency of PE cells into mature IPCs via paracrine effects.

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Acknowledgments

This research was supported by grants from the Samsung Biomedical Research Institute (GL1B30811) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare (A084065) & (HI12C1853).

Conflict of Interest

The authors indicate no potential conflicts of interest.

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Authors and Affiliations

  1. Division of Endocrinology and Metabolism, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, Republic of Korea

    Bea Jun Oh, Seung-Hoon Oh, Jin Myung Choi, Sang-Man Jin, Woo-Young Shim, Myung-Shik Lee, Moon-Kyu Lee & Jae Hyeon Kim

  2. Samsung Biomedical Research Institute, Seoul, Republic of Korea

    Bea Jun Oh

  3. Department of Endocrinology and Metabolism, Gachon University Gil Medical Center, Incheon, Republic of Korea

    Kwang-Won Kim

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  1. Bea Jun Oh
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Fig. S1

Expression of SOX17 and FOXA2 according to Activin A concentration. (a) and (b) Q-PCR analyses of SOX17 and FOXA2. (C) Expression of β-cell-specific gene markers according to the Activin A concentration at the final stage of differentiation (stage 4). (PPTX 117 kb)

Fig. S2

Effect of LY294002 on differentiation of definitive endoderm. Differentiated mES cells after LY294002 treatment increased expression of definitive endoderm markers (SOX17 and FOXA2), as detected by RT-PCR (a) and immunofluorescence (b). Scale bar: 100 μm. (PPTX 561 kb)

Fig. S3

Effect of ILV on differentiation of insulin-producing cells. After adding ILV+FGF10, the expression of islet genes improved after the final induction compared with expression without ILV+FGF10 treatment, as detected by RT-PCR. (PPTX 313 kb)

Fig. S4

Characterization of hiPS-derived differentiation. Cells were induced at the corresponding stages. (a) Flow chart of differentiation protocol. (b) Q-PCR showing a transient expression of the mesendoderm marker T (BRACHYURY homolog) on day 1, followed by SOX17 and FOXA2 expression on day 3. Expression of SOX17 and OCT4 decreased progressively. (c) Immunofluorescence analysis of SOX17 and FOXA2 at the definitive endoderm. Scale bar: 50 μm. (d) Primitive gut-tube markers HNF-1β, HNF4, and FOXA2 were detected at stage 2. Scale bar: 100 μm. Inserts, higher magnification of HNF-1β-, HNF4α- and FOXA2-expressing cells. (e) Fluorescence photographs showing expression of the indicated markers for posterior foregut (HNF6, SOX9 and PDX-1), endocrine precursors (NKX6.1 and PAX6), and insulin-producing cells (C-peptide and insulin), Scale bar: 100 um. Right panels, higher magnifications. (f) Q-PCR was performed using RNA samples from differentiated cells collected at the indicated stages to quantify the levels of PDX-1, NKX6.1, and insulin gene expression. The level of expression at each stage was normalized to undifferentiated hiPS. (PPTX 552 kb)

(PPTX 582 kb)

Fig. S5

Gene expression of differentiated cells at each stage. (a) hiPS cells were differentiated into insulin-producing cells as described above. Cell samples were collected at each stage and analyzed by Q-PCR for HNF1β, HNF4α, HLXB9, PTF1a, NGN3, NKX2.2, GCK and ALB. For each sample, relative expression levels were normalized to the hiPS level, which was set as 1. (b) Overview of temporal gene expression during differentiation from hiPS. For each gene, the level of expression at each time point was normalized to the maximal level, which was set as 1. Each graph represents the trend over time. (PPTX 218 kb)

Fig. S6

Immunostaining for insulin (red) and glucagon (green) at day 30 (PPTX 192 kb)

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Oh, B.J., Oh, SH., Choi, J.M. et al. Co-culture with Mature Islet Cells Augments the Differentiation of Insulin-Producing Cells from Pluripotent Stem Cells. Stem Cell Rev and Rep 11, 62–74 (2015). https://doi.org/10.1007/s12015-014-9554-8

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